Target - FabG 3-oxoacyl-(acyl-carrier protein) reductase (P. falciparum)

Dr. B Notes (092413):


Valdiation dock againt main ACP site
get ~15 positive and negative control ligands
Use 'similar' FabG PDB structures with ligands
Use Binding DB
get 3-D compounds from PubChem
negative controls: aspirin, or go to Chembridge Hit2Lead and get some random structures (only can get as 2-D files - but LigPrep will convert to 3D)

concatenate SDF files of control ligands
go to LigPrep with all Control Ligands




Validation dock of NADPH site
- have gold remove and re-dock NADPH into same pocket
- inspect the RMS value

• 
  
• Target (protein/gene name): FabG 3-oxoacyl-(acyl-carrier protein)reductase
  
  
• NCBI Gene # or RefSeq#: XM_001352064
  
  
• Protein ID (NP or XP #) or Wolbachia #: XP_001352100
  
  
• Organism (including strain): Plasmodium falciparum
  
  
• Etiologic Risk Group (see link below): 2
  
  
• Background/Disease Information:
  Plasmodium falciparum causes malaria with the highest rates of mortality. More than three out of four cases of malaria are caused by Plasmodium falciparum, and almost every death from malaria resuts from this parasite.
  Choloroquine was the most efficient method of combating malaria, however, resistance to the drug has begun to develop. Additionally, resistance to sulfadoxine-pyrimethamine has begun to spread into East Africa from South-East Asia. As a result, there is a need to explore possible drugs to combat malaria.
  
  
• Essentiality of this protein:
  
  Gene/Ortholog: Tb927.2.5210 (OG4_10098); Phenotype: significant gain of fitness in bloodstream forms (3 days); Source study: alsford
  Gene/Ortholog: Tb927.2.5210 (OG4_10098); Phenotype: no significant loss or gain of fitness in bloodstream forms (6 days); Source study: alsford
  Gene/Ortholog: Tb927.2.5210 (OG4_10098); Phenotype: significant gain of fitness in procyclic forms; Source study: alsford
  Gene/Ortholog: Tb927.2.5210 (OG4_10098); Phenotype: no significant loss or gain of fitness in differentiation of procyclic to bloodstream forms; Source study: alsford
  Gene/Ortholog: Tb927.10.11930 (OG4_10098); Phenotype: significant gain of fitness in bloodstream forms (3 days); Source study: alsford
  Gene/Ortholog: Tb927.10.11930 (OG4_10098); Phenotype: significant loss of fitness in bloodstream forms (6 days); Source study: alsford
  Gene/Ortholog: Tb927.10.11930 (OG4_10098); Phenotype: no significant loss or gain of fitness in procyclic forms; Source study: alsford
  Gene/Ortholog: Tb927.10.11930 (OG4_10098); Phenotype: significant loss of fitness in differentiation of procyclic to bloodstream forms; Source study: alsford
  
  
• Complex of Protein?: No
  
  
• Druggable Target: 0.6 (yes)
  
  
• EC #: 1.1.1.100
  
  
• Link to BRENDA EC # page: http://www.brenda-enzymes.org/php/result_flat.php4?ecno=1.1.1.100
  
  
• Enzyme Assay information: NADPH
  • "The functional activity of the refolded protein was monitored by the decrease in absorbance at 340 nm due to oxidation of NADPH to NADP(+) in the presence of the substrate, acetoacetyl-CoA. The standard reaction mixture in a final volume of 100 μl contained 10 mM Tris–Cl, pH 7.5, 18 μg pure protein, and 250 μM acetoacetyl-CoA. The reaction was started by the addition of 125 μM NADPH."
    • http://www.sciencedirect.com/science/article/pii/S1046592805000604
  • "The optimal ionic strength for the reaction was determined in 20 mM Hepes, pH 7.4, 200 μM NADPH, 50 mM AcAcNAC and various concentrations of NaCl (0–750 mM) using 4.5 μg of OAR. The pH optimum for the reaction was determined using a mixed buffer system consisting of 50 mM Mes, 50 mM Ches [2-(N-cyclohexylamino)ethanesulphonic acid] and 50 mM Hepes at a pH range of 5–10, 200 μM NADPH, 50 mM AcAcNAC and 2.4 μg of OAR. The ionic strength was maintained at 0.325 M with NaCl.
    The standard assay for OAR contained 50 mM sodium phosphate buffer, pH 6.8, containing 0.25 M NaCl, 200 μM NADPH, 50 mM AcAcNAC or 0.5 mM AcAcCoA and 0.2–0.8 μg of OAR in a final volume of 0.5 ml. The assay mixture was pre-incubated for 5 min at 25 °C before the reaction was initiated by the addition of substrate or enzyme. The initial rate of NADPH oxidation was determined spectrophotometrically, by monitoring the reduction in absorbance at 340 nm (Beckman DU 640 or Shimadzu UV-1601 spectrometer). Under these standard assay conditions, activity was proportional to enzyme added between 0.09 and 14 μg of OAR."
    • http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1360695/
      
      
• Structure Available (PDB or Homology model): 2C07
  
  
• Current Inhibitors: 8305 known inhibitors
  
  
• Amino Acid Sequence:
  • MSVLHRFYLFFLFTKFFHCYKISYVLKNAKLAPNHAIKNINSLNLLSENKKENYYYCGEN KVALVTGAGRGIGREIAKMLAKSVSHVICISRTQKSCDSVVDEIKSFGYESSGYAGDVSK KEEISEVINKILTEHKNVDILVNNAGITRDNLFLRMKNDEWEDVLRTNLNSLFYITQPIS KRMINNRYGRIINISSIVGLTGNVGQANYSSSKAGVIGFTKSLAKELASRNITVNAIAPG FISSDMTDKISEQIKKNIISNIPAGRMGTPEEVANLACFLSSDKSGYINGRVFVIDGGLSP
    
    
• Length of your protein in Amino Acids: 301 amino acids
  
  
• Molecular Weight of your protein in kiloDaltons using Expasy ProtParam website: 33347.3 g/mol
  
  
• TMpred graph Image: (input amino acid sequence)
  • 
    
    
• CDS Gene sequence: 906 bases
  • ATGAGTGTTCTACATAGATTTTATCTATTTTTCTTGTTTACAAAGTTCTTCCATTGTTAT AAAATTTCTTATGTATTAAAAAATGCAAAGCTAGCTCCCAACCATGCAATTAAAAATATT AATTCATTAAATTTATTGTCTGAAAATAAGAAGGAAAACTATTATTATTGTGGGGAAAAT AAAGTTGCTTTAGTAACAGGTGCAGGAAGAGGAATAGGTAGAGAGATCGCTAAAATGTTG GCAAAATCAGTATCTCATGTTATATGTATAAGTAGAACGCAAAAATCATGTGATAGTGTT GTTGATGAAATAAAATCATTTGGTTATGAATCATCAGGTTATGCAGGTGATGTATCTAAA AAAGAAGAAATAAGTGAAGTGATTAATAAAATTTTGACAGAACATAAAAATGTAGATATA TTAGTTAATAATGCTGGAATAACTAGAGATAATCTTTTTCTAAGGATGAAAAATGATGAA TGGGAAGATGTGTTAAGGACAAATTTAAATTCTCTATTTTATATAACACAACCTATATCA AAAAGAATGATTAATAATAGATATGGTCGAATAATTAATATATCAAGTATAGTAGGGTTA ACAGGAAATGTAGGACAAGCAAATTATTCTTCATCGAAAGCTGGTGTTATTGGTTTTACA AAAAGCTTAGCAAAAGAATTAGCTTCAAGAAATATAACTGTAAATGCCATAGCCCCTGGA TTTATATCTAGTGATATGACAGATAAAATTAGCGAACAAATAAAGAAGAACATAATTTCA AACATTCCTGCTGGACGAATGGGAACACCAGAAGAAGTAGCTAATTTAGCTTGTTTTTTA TCATCAGATAAGTCTGGTTATATTAATGGTCGAGTTTTCGTAATAGACGGTGGACTATCACCTTAA
    
    
• GC % content: 28.91%
  
  
• CDS gene sequence codon optimized:
  • ATGTCTGTACTCCACCGCTTTTACCTCTTTTTCCTGTTCACCAAATTCTTCCACTGCTAC AAGATCTCCTACGTTCTCAAAAATGCGAAGCTGGCGCCGAACCACGCGATTAAGAACATC AACTCTCTGAACCTGCTGTCTGAAAATAAGAAAGAAAACTATTACTACTGCGGTGAAAAC AAGGTTGCGCTGGTCACTGGTGCAGGTCGTGGTATTGGTCGCGAGATTGCGAAAATGCTG GCGAAGTCTGTTTCTCATGTGATCTGCATCTCTCGTACCCAAAAATCTTGCGACTCTGTT GTGGACGAGATCAAGTCTTTCGGTTACGAATCTTCTGGCTACGCGGGTGACGTTTCCAAA AAGGAGGAGATTTCCGAAGTTATCAACAAAATCCTGACCGAACACAAGAACGTTGACATC CTCGTTAACAATGCGGGTATCACTCGTGATAATCTGTTCCTGCGTATGAAAAACGACGAA TGGGAAGACGTTCTGCGTACCAACCTGAACTCCCTGTTCTACATCACTCAGCCGATCTCT AAACGTATGATTAACAACCGCTATGGTCGCATCATCAACATCTCTTCTATCGTTGGCCTG ACTGGTAACGTTGGTCAAGCTAACTACTCTTCTTCTAAAGCGGGTGTTATTGGTTTTACC AAGTCTCTGGCCAAAGAACTGGCGTCTCGTAACATCACCGTTAATGCAATCGCGCCAGGT TTCATCTCTAGCGACATGACCGATAAGATTTCTGAACAAATCAAGAAAAACATCATTTCT AACATTCCGGCTGGCCGTATGGGCACCCCGGAAGAAGTTGCCAACCTCGCGTGCTTCCTG TCTTCTGATAAATCTGGTTACATCAATGGCCGCGTTTTCGTTATCGACGGTGGTCTGTCTCCGTAA
• GC % Content for gene (codon optimized): 46.13%
  
  
• Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
  (Link:https://drive.google.com/#folders/0B4O2KqKh2q_-UnZrYzVIWlhRZVU)
  • Forward Primer
    • 5’ TAC TTC CAA TCC ATG TCT GTA CTC CAC CGC TTT TA 3’ 35 bp
      
      
  • Reverse Primer
    • 5’ ACG GTG GTC TGT CTC CGT AAC AGT AAA GGT GGA TA 3’
    • Reverse Complement
      • 5’ TAT CCA CCT TTA CTG TTA CGG AGA CAG ACC ACC GT 3’ 35 bp